Poster A1-1936 Mailing address: a New Oxazolidinone, in ... al...Poster A1-1936 Background. S....
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ABSTRACT METHODS REFERENCES BACKGROUND AND AIM 49 th ICAAC San Francisco, CA, USA September 12-15, 2009 Poster A1-1936 Background. S. aureus invades and persists within a variety of non-phagocytic cells. This may play a determining role in relapsing diseases like skin or respiratory tract infections, osteomyelitis, and endocarditis. Effective treatment may require drug accumulation in target cells. We have previously shown that radezolid (RDZ) accumulates 8-fold in human macrophages (Lemaire et al. ECCMID 2009, O29). We have now determined its intracellular accumulation and activity in a series of relevant non-phagocytic cells. Methods. Cells are listed in the table. Cellular concentration was measured at 24h using 14 C-RDZ, and accumulation calculated using a mean cell volume of 5 μl/mg cell prot. MICs and dose-effect relationships at 24h were determined as described (Barcia-Macay et al, AAC, 2006), using MRSA SA238 (clinical isolate) and a linezolid (LZD) R in vitro mutant thereof (SA238L). Results are expressed as the change in 24h inoculum compared to t=0. Results. The cellular accumulation of RDZ and its intracellular activity (against S. aureus strain SA238, [MICs: 0.5-1 and 2 mg/L for RDZ and LZD] was similar in all cell types. Against the LZD R mutant (MICs: 2 and 8 mg/L for RDZ and LZD), RDZ C static and E max remained constant while LZD activity was diminished (C static range from 15.7 mg/L in Calu-3 to > 100 mg/L in osteoblasts; E max , –0.6 log in keratinocytes to +0.5 log in osteoblasts). Conclusions. RDZ accumulates in non-phagocytic cells to the same extent as in macrophages and shows comparable intracellular activity against both LZD S and LZD R MRSA. LZD was systematically less effective against the resistant strain. It is now well accepted that Staphylococcus aureus is a facultative intracellular pathogen and that its capacity to survive within non-phagocytic cells may play an important role in the persistent and recurrent character of several in- fections, such as complicated skin and soft tissue infections, osteomyelitis, endocarditis, or pulmonary infections associated with cystic fibrosis (1). Effective treatment of such infections may therefore require the use of antibi- otics accumulating intracellularly and demonstrating activity therein. In this context, we have shown that radezolid (RDZ), a novel oxazolidinone antibiotic (see structure in Figure 1, and description on poster A1-1937), markedly accumulates (approx. 10-fold) within phagocytic cells (PMN and macrophages) and shows improved potency towards both linezolid- susceptible and linezolid-resistant S. aureus phagocytized by human THP-1 macrophages (2; A1-1937). The aim of the present study was to measure the intracellular accumulation and activity of radezolid in a series of non-phagocytic cells originating from the tissues subject to persistent infections. CONCLUSIONS Bacteria and susceptibility testings. S. aureus SA238 and SA238L, a linezolid- resistant mutant thereof selected in vitro, were used in the present study (3). MICs were determined in Mueller Hinton Broth as previously described (4). Cell lines. Experiments were conducted with primary cultures of rat fibroblasts or of human skin keratinocytes, and with continous cell lines of human lung epi- thelial cells (ATCC, Manassas, VA), normal human osteoblasts (Lonza), and human umbilical vein endothelial cells (HUVEC, Lonza). Determination of the cellular accumulation. The cellular content of radezolid was determined by scintillation, cells having been incubated with 14 C-RDZ. Cell infection and assessment of the intracellular activities of antibiotics. Infection of cells was performed exactly as described before (3-5) with typical starting inoculum of ~ 1 to 2 x10 6 CFU/mg prot. Antibiotic activity was examined after 24 h in the presence of increasing concentrations of antibiotics (0.01- to 100 mg/L), and results were expressed as the change in the inoculum at 24 h com- pared to time 0 (4-5). Data were analyzed by non-linear regression using Hill's equation to calculate pharmacological descriptors (E max , maximal reduction of the intracellular inoculum [in log 10 units] for an infinitely large antibiotic concen- tration; EC 50 , antibiotic concentration [in mg/L] yielding a response half-way be- tween E 0 and E max , as obtained from the Hill equation), together with the static concentration (C static ) estimated by graphical interpolation. Accumulation and Intracellular Activity of Radezolid, a New Oxazolidinone, in non-Phagocytic Cells S. Lemaire 1 , K. Kosowska-Shick 2 , P. C. Appelbaum 2 , J.-P. Pirnay 3 , G. Verween 3 , P. De Corte 3 , D. De Vos 3 , P. M. Tulkens 1 and F. Van Bambeke 1 1 Université catholique de Louvain, Brussels, Belgium; 2 Hershey Medical Center, Hershey, PA, USA; 3 Queen Astrid Military Hospital, Brussels, Belgium • • -2 -1 0 1 2 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Fibroblasts -2 -1 0 1 2 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Keratinocytes -2 -1 0 1 2 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Osteoblasts -2 -1 0 1 2 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 HUVEC -2 -1 0 1 2 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 LZD (sa 238) Radezolid (sa 238) Calu-3 LZD (sa 238 L) Radezolid (sa 238 L) MIC = 2 mg/L MIC = 0.5 mg/L MIC = 16 mg/L MIC = 2 mg/L log 10 extracellular concentration (mg/L) ∆ log CFU from time 0 Figure 2. Concentration-killing effect of linezolid vs. radezolid against linezolid- susceptible SA238 and linezolid-resistant (SA238L) strains phagocytosed by differ- ent cell types. Cells were incubated with antibiotics for 24 h in the presence of in- creasing concentrations of antibiotics. The graph shows the change in CFU per mg of cell protein. Radezolid shows similar efficacy (E max ) and potency (C static ) against both the SA238 suscep- tible and the SA238L resistant strains. Radezolid is 3.4- to 6.3-fold more potent than linezolid against the SA238 susceptible strain and 8.7 to > 100 fold against the SA238L resistant strain. Although MICs are the same (2 mg/L) , radezolid shows 2-3-fold higher potency against the resistant strain SA238L than linezolid against the susceptible strain SA238. Radezolid shows similar efficacy in all cell types; linezolid efficacy is reduced against the SA238L resistant strain in all cells. At 24 h, radezolid accumulates to a high and similar level within all non-phagocytic cells, reaching an accumulation factor similar to that observed within phagocytic cells (2). RESULTS Radezolid accumulates to a similar extent in all cell types. Radezolid shows globally a similar efficacy (E max ) relative to linezolid, but a higher relative po- tency (lower C static ) than linezolid against intracellular S. aureus in all cell types. Radezolid shows similar intracellular activities (E max and C static ) against linezolid-susceptible and linezolid resistant strains, suggesting that it could be a useful substitute to linezolid to treat in- tracellular infections caused by these resistant bacteria. (1) Garzoni & Kelley. Trends Microbiol. (2009) 17:59-65. (2) Lemaire et al. 26th ICC, Toronto, ON (2009) (3) Kosowska-Shick et al. Antimicrob. Agents Chemother. (2006) 50:765-769. (4) Barcia-Macay et al. Antimicrob. Agents Chemother. (2006) 50: 841-851. (5) Lemaire et al, Antimicrob. Agents Chemother. (2008) 52: 2797-2805. This poster will be made available for download after the meeting: http://www.facm.ucl.ac.be/posters.htm We thank M.C. Cambier and C. Misson for technical assistance. S.L. and F.V.B. are Post-doctoral and Senior Research Associate of the Belgian Fonds de la Re- cherche Scientifique (FSR-FNRS). This work was supported by the Belgian Fonds de la Recherche Scientifique Médicale (FRSM) and a grant-in-aid from Rib-X Pharmaceuticals Inc. • • a Static concentration : concentration yielding no apparent change in the inoculum compared to time 0 b Maximal Efficacy: Change in log10 CFU compared to time 0 for an infinitely large concentration of antibiotic Mailing address: F. Van Bambeke UCL 73.70 av. Mounier 73, 1200 Brussels, Belgium [email protected] • • • Cell lines cellular accumulation (24h) Rat fibroblasts (primary culture) 10.9 ± 2.1 Human skin keratinocytes (primary cultures) 16.1 ± 2.0 Normal human osteoblasts (NHost) 9.7 ± 0.4 Human Umbilical Vein endothelial cells (HUVEC) 10.5 ± 0.1 Human lung epithelial cells (Calu-3) 8.3 ± 1.0 Cellular accumulation of radezolid log 10 extracellular concentration (mg/L) Intracellular activity of antibiotics Linezolid O N O N O O F N H Radezolid N N N N O O F N O H N H H Figure 1 C static a (mg/L) E max b C static a (mg/L) E max b Fibroblasts ~ 6.6 -0.8 ± 0.1 ~ 1.5 -1.5 ± 0.3 4.4 Keratinocytes ~ 9.5 -1.2 ± 0.3 ~ 1.5 -1.2 ± 0.1 6.3 Osteoblasts ~ 6.8 -1.2 ± 0.2 ~ 1.3 -1.3 ± 0.1 5.2 HUVEC ~ 5.8 -0.5 ± 0.1 ~ 1.7 -0.7 ± 0.1 3.4 Calu-3 ~ 2.6 -1.2 ± 0.2 ~ 0.6 -1.3 ± 0.1 4.3 Fibroblasts ~ 42 -0.2 ± 0.1 ~ 1.9 -1.2 ± 0.1 22 Keratinocytes ~ 27 -0.6 ± 0.3 ~ 3.3 -1.3 ± 0.2 8.2 Osteoblasts > 100 +0.5 ± 0.1 ~ 2.8 -1.0 ± 0.1 > 35 HUVEC ~ 20 -0.4 ± 0.1 ~ 2.3 -0.6 ± 0.1 8.7 Calu-3 ~ 15 -0.2 ± 0.2 ~ 0.6 -1.4 ± 0.1 25 strain Cell line linezolid radezolid Cstatic (LZD/RDZ ratio) Pharmacological descriptors as calculated by non-linear (sigmoid) regresion of the dose-response curves illustrated in Figure 2. SA238 (LZD-S) SA238L (LZD-R)
Transcript of Poster A1-1936 Mailing address: a New Oxazolidinone, in ... al...Poster A1-1936 Background. S....
ABSTRACT
METHODS
REFERENCES
BACKGROUND AND AIM
49th ICAAC San Francisco, CA, USASeptember 12-15, 2009
Poster A1-1936
Background. S. aureus invades and persists within a variety of non-phagocytic cells. This may play a determining role in relapsing diseases like skin or respiratory tract infections, osteomyelitis, and endocarditis. E�ective treatment may require drug accumulation in target cells. We have previously shown that radezolid (RDZ) accumulates 8-fold in human macrophages (Lemaire et al. ECCMID 2009, O29). We have now determined its intracellular accumulation and activity in a series of relevant non-phagocytic cells.
Methods. Cells are listed in the table. Cellular concentration was measured at 24h using 14C-RDZ, and accumulation calculated using a mean cell volume of 5 µl/mg cell prot. MICs and dose-e�ect relationships at 24h were determined as described (Barcia-Macay et al, AAC, 2006), using MRSA SA238 (clinical isolate) and a linezolid (LZD)R in vitro mutant thereof (SA238L). Results are expressed as the change in 24h inoculum compared to t=0.Results. The cellular accumulation of RDZ and its intracellular activity (against S. aureus strain SA238, [MICs: 0.5-1 and 2 mg/L for RDZ and LZD] was similar in all cell types. Against the LZDR mutant (MICs: 2 and 8 mg/L for RDZ and LZD), RDZ Cstatic and Emax remained constant while LZD activity was diminished (Cstatic range from 15.7 mg/L in Calu-3 to > 100 mg/L in osteoblasts; Emax, –0.6 log in keratinocytes to +0.5 log in osteoblasts).Conclusions. RDZ accumulates in non-phagocytic cells to the same extent as in macrophages and shows comparable intracellular activity against both LZDS and LZDR MRSA. LZD was systematically less e�ective against the resistant strain.
It is now well accepted that Staphylococcus aureus is a facultative intracellular pathogen and that its capacity to survive within non-phagocytic cells may play an important role in the persistent and recurrent character of several in-fections, such as complicated skin and soft tissue infections, osteomyelitis, endocarditis, or pulmonary infections associated with cystic �brosis (1). E�ective treatment of such infections may therefore require the use of antibi-otics accumulating intracellularly and demonstrating activity therein. In this context, we have shown that radezolid (RDZ), a novel oxazolidinone antibiotic (see structure in Figure 1, and description on poster A1-1937), markedly accumulates (approx. 10-fold) within phagocytic cells (PMN and macrophages) and shows improved potency towards both linezolid-susceptible and linezolid-resistant S. aureus phagocytized by human THP-1 macrophages (2; A1-1937). The aim of the present study was to measure the intracellular accumulation and activity of radezolid in a series of non-phagocytic cells originating from the tissues subject to persistent infections.
CONCLUSIONS
Bacteria and susceptibility testings. S. aureus SA238 and SA238L, a linezolid-resistant mutant thereof selected in vitro, were used in the present study (3). MICs were determined in Mueller Hinton Broth as previously described (4).
Cell lines. Experiments were conducted with primary cultures of rat �broblasts or of human skin keratinocytes, and with continous cell lines of human lung epi-thelial cells (ATCC, Manassas, VA), normal human osteoblasts (Lonza), and human umbilical vein endothelial cells (HUVEC, Lonza).
Determination of the cellular accumulation. The cellular content of radezolid was determined by scintillation, cells having been incubated with 14C-RDZ.
Cell infection and assessment of the intracellular activities of antibiotics. Infection of cells was performed exactly as described before (3-5) with typical starting inoculum of ~ 1 to 2 x106 CFU/mg prot. Antibiotic activity was examined after 24 h in the presence of increasing concentrations of antibiotics (0.01- to 100 mg/L), and results were expressed as the change in the inoculum at 24 h com-pared to time 0 (4-5). Data were analyzed by non-linear regression using Hill's equation to calculate pharmacological descriptors (Emax, maximal reduction of the intracellular inoculum [in log10 units] for an in�nitely large antibiotic concen-tration; EC50 , antibiotic concentration [in mg/L] yielding a response half-way be-tween E0 and Emax, as obtained from the Hill equation), together with the static concentration (Cstatic) estimated by graphical interpolation.
Accumulation and Intracellular Activity of Radezolid, a New Oxazolidinone, in non-Phagocytic Cells
S. Lemaire1, K. Kosowska-Shick2, P. C. Appelbaum2, J.-P. Pirnay3 , G. Verween3, P. De Corte3, D. De Vos3, P. M. Tulkens1 and F. Van Bambeke1
1 Université catholique de Louvain, Brussels, Belgium; 2 Hershey Medical Center, Hershey, PA, USA; 3 Queen Astrid Military Hospital, Brussels, Belgium
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MIC = 2 mg/L
MIC = 0.5 mg/L
MIC = 16 mg/L
MIC = 2 mg/L
log10 extracellular concentration (mg/L)
∆ lo
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Figure 2. Concentration-killing e�ect of linezolid vs. radezolid against linezolid-susceptible SA238 and linezolid-resistant (SA238L) strains phagocytosed by di�er-ent cell types. Cells were incubated with antibiotics for 24 h in the presence of in-creasing concentrations of antibiotics. The graph shows the change in CFU per mg of cell protein.
Radezolid shows similar e�cacy (Emax) and potency (Cstatic) against both the SA238 suscep-tible and the SA238L resistant strains.
Radezolid is 3.4- to 6.3-fold more potent than linezolid against the SA238 susceptible strain and 8.7 to > 100 fold against the SA238L resistant strain.
Although MICs are the same (2 mg/L) , radezolid shows 2-3-fold higher potency against the resistant strain SA238L than linezolid against the susceptible strain SA238.
Radezolid shows similar e�cacy in all cell types; linezolid e�cacy is reduced against the SA238L resistant strain in all cells.
At 24 h, radezolid accumulates to a high and similar level within all non-phagocytic cells, reaching an accumulation factor similar to that observed within phagocytic cells (2).
RESULTS
Radezolid accumulates to a similar extent in all cell types.
Radezolid shows globally a similar e�cacy (Emax) relative to linezolid, but a higher relative po-tency (lower Cstatic) than linezolid against intracellular S. aureus in all cell types.
Radezolid shows similar intracellular activities (Emax and Cstatic) against linezolid-susceptible and linezolid resistant strains, suggesting that it could be a useful substitute to linezolid to treat in-tracellular infections caused by these resistant bacteria.
(1) Garzoni & Kelley. Trends Microbiol. (2009) 17:59-65.(2) Lemaire et al. 26th ICC, Toronto, ON (2009)(3) Kosowska-Shick et al. Antimicrob. Agents Chemother. (2006) 50:765-769.(4) Barcia-Macay et al. Antimicrob. Agents Chemother. (2006) 50: 841-851.(5) Lemaire et al, Antimicrob. Agents Chemother. (2008) 52: 2797-2805.
This poster will be made available for download after the meeting: http://www.facm.ucl.ac.be/posters.htm
We thank M.C. Cambier and C. Misson for technical assistance. S.L. and F.V.B. are Post-doctoral and Senior Research Associate of the Belgian Fonds de la Re-cherche Scienti�que (FSR-FNRS). This work was supported by the Belgian Fonds de la Recherche Scienti�que Médicale (FRSM) and a grant-in-aid from Rib-X Pharmaceuticals Inc.
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a Static concentration : concentration yielding no apparent change in the inoculum compared to time 0b Maximal E�cacy: Change in log10 CFU compared to time 0 for an in�nitely large concentration of antibiotic
Mailing address: F. Van Bambeke UCL 73.70 av. Mounier 73,1200 Brussels, [email protected]
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Cell linescellular accumulation
(24h)
Rat �broblasts (primary culture) 10.9 ± 2.1
Human skin keratinocytes (primary cultures) 16.1 ± 2.0
Normal human osteoblasts (NHost) 9.7 ± 0.4
Human Umbilical Vein endothelial cells (HUVEC) 10.5 ± 0.1
Human lung epithelial cells (Calu-3) 8.3 ± 1.0
Cellular accumulation of radezolid
log10 extracellular concentration (mg/L)
Intracellular activity of antibiotics
Linezolid
O
NO NO
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NH
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H Figure 1
Cstatica (mg/L) Emaxb Cstatica
(mg/L) Emaxb
Fibroblasts ~ 6.6 -0.8 ± 0.1 ~ 1.5 -1.5 ± 0.3 4.4
Keratinocytes ~ 9.5 -1.2 ± 0.3 ~ 1.5 -1.2 ± 0.1 6.3
Osteoblasts ~ 6.8 -1.2 ± 0.2 ~ 1.3 -1.3 ± 0.1 5.2
HUVEC ~ 5.8 -0.5 ± 0.1 ~ 1.7 -0.7 ± 0.1 3.4
Calu-3 ~ 2.6 -1.2 ± 0.2 ~ 0.6 -1.3 ± 0.1 4.3
Fibroblasts ~ 42 -0.2 ± 0.1 ~ 1.9 -1.2 ± 0.1 22
Keratinocytes ~ 27 -0.6 ± 0.3 ~ 3.3 -1.3 ± 0.2 8.2
Osteoblasts > 100 +0.5 ± 0.1 ~ 2.8 -1.0 ± 0.1 > 35
HUVEC ~ 20 -0.4 ± 0.1 ~ 2.3 -0.6 ± 0.1 8.7
Calu-3 ~ 15 -0.2 ± 0.2 ~ 0.6 -1.4 ± 0.1 25
strain Cell linelinezolid radezolid Cstatic
(LZD/RDZ ratio)
Pharmacological descriptors as calculated by non-linear (sigmoid) regresion of the dose-response curves illustrated in Figure 2.
SA238 (LZD-S)
SA238L (LZD-R)
